Prognostic Role of Osteopontin Expression in Malignant Pleural Mesothelioma

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1 Anatomic Pathology / Prognostic Role of Osteopontin in Mesothelioma Prognostic Role of Osteopontin Expression in Malignant Pleural Mesothelioma Susanna Cappia, 1 Luisella Righi, MD, PhD, 1 Dario Mirabelli, MD, 2 Paolo Ceppi, 1 Elisa Bacillo, 1 Francesco Ardissone, MD, 1 Luca Molinaro, MD, 1 Giorgio V. Scagliotti, MD, 1 and Mauro Papotti, MD 1 Key Words: Immunohistochemistry; Mesothelioma; Osteopontin; Pleura; Prognosis DOI: /TWCQV536WWRNEU51 Abstract Malignant pleural mesothelioma (MPM) represents highly aggressive neoplasms with a mean survival of approximately 10 months. Osteopontin, a glycoprotein involved in cell-matrix interactions correlated with invasion and metastatic spread in several tumors, has recently been proposed as a serum marker of MPM in asbestos-exposed subjects. The aim of this study was to define the prognostic role of osteopontin in MPM. For the study, 32 long-term survivors (>24 months) and a random sample of 69 short-term survivors ( 24 months) were matched according to the main clinicopathologic features. Immunohistochemical osteopontin expression in tissue specimens was quantified through the HScore (histologic scoring) method and correlated with clinicopathologic parameters and survival. Osteopontin expression was significantly lower in long-term compared with short-term survivors (P <.0001), and overall survival analysis showed that low osteopontin expression was associated with longer survival; multivariate analysis confirmed the value of osteopontin expression as an independent prognostic factor (P <.0001). Malignant pleural mesothelioma (MPM) is a highly lethal tumor associated in the vast majority of the cases with asbestos exposure. The disease is largely unresponsive to conventional chemotherapy or radiotherapy, and surgical treatment (extrapleural pneumonectomy or pleurectomy) has not shown a significant survival benefit compared with supportive treatment. 1 A multimodal approach to improve survival in selected patients remains controversial, and the definition of relevant prognostic factors is lacking. 2 Overall, the median survival time is largely dependent on the tumor stage at diagnosis 3 and in unresectable disease does not exceed 11 months. 4 Long-term survival (>2 years) of patients with MPMs has rarely been reported. 3 Osteopontin is an acidic, calcium-binding glycol-phosphoprotein of 44 to 66 kda, depending on species and cell types. It is found in all body fluids and secreted by osteoclasts, macrophages, cardiac fibroblasts, and activated T cells. 5 Osteopontin binds multiple integrins, including α v β 1, α v β 3, and α v β 5, and it may also be a ligand for some forms of CD44, especially variants 6 and 7. 6,7 Many cellular processes, including cell attachment, spreading and migration, homing of lymphocytes and other hematopoietic cells, and vascular remodeling, are thought to involve osteopontin binding to cell surface receptors. 8,9 Osteopontin is overexpressed in many human tumors, including breast, 10 lung, 11 ovarian, 12 and gastric 13 tumors; hepatocellular carcinomas 14 ; and prostate tumors, 15 and it was proposed as a potential prognostic marker in many of these neoplasms. Osteopontin immunoreactivity is absent in normal mesothelial cells, and it is weakly expressed in reactive mesothelial proliferations. 16,17 Recently, in asbestos-exposed people, high serum levels of osteopontin were correlated with the presence of MPM but not pleural plaques or interstitial lung disease. 16,18 58 Am J Clin Pathol 2008;130: DOI: /TWCQV536WWRNEU51

2 Anatomic Pathology / Original Article The aims of the present study were to investigate the immunohistochemical osteopontin levels in a large series of MPMs and to correlate the results with clinicopathologic parameters. We show that osteopontin reactivity is significantly reduced in long-surviving patients with mesothelioma, expanding its known prognostic role to mesothelial malignancies. Materials and Methods Case Selection All 394 cases diagnosed at the University of Turin Division of Pathology St Luigi Hospital, Turin, Italy, between 1989 and 2003 were identified. All patients were followed up until death or for at least 2 years. The study was limited to data for all patients with survival of more than 24 months (longterm survivors; LS group) and a random sample of patients with survival of less than 24 months (short-term survivors, SS group). Based on follow-up data provided by the Regional Mesothelioma Registry, 70 LS cases were identified. After case review by 2 pathologists (M.P. and L.R.) on H&E-, mucin- (periodic acid Schiff with and without diastase treatment), carcinoembryonic antigen, and calretinin-stained slides, 32 cases from the LS group were retained for the purpose of the study according to the revised histologic diagnosis and available histopathologic (but not cytologic) tissue material; 38 cases were excluded owing to the presence of a cytologic diagnosis only, absent or insufficient archival material in paraffin blocks, or change in the original diagnosis. The SS group was created by randomly selecting from the same database 69 patients with survival of 24 months or less, as commonly observed in most cases, and matched (at least 2 for each LS case) according to sex, age, asbestos exposure (presence, level, type [ie, occupational, household, or environmental], and duration), stage of the disease at thoracoscopy (ie, visceral pleural involvement and presence of vegetating lesions), and histologic tumor type (World Health Organization criteria 19 ). Clinicopathologic data, which were previously prospectively obtained at the Thoracic Surgery Division, St Luigi Hospital, included smoking history, symptoms (cough, dyspnea, and chest pain), latency of development of disease (intended as time between start of exposure and diagnosis), weight loss, performance status, time from symptoms to diagnosis, blood cell count, and treatment administered. All patients underwent talc pleurodesis. A further group of 81 consecutive cases of MPM (CC group) diagnosed at the same institution from January 2005 to June 2007 by thoracoscopic biopsies was also studied for osteopontin expression. Prevalence data of osteopontin reactivity in such unselected cases were compared with those for the LS and SS groups. Finally, pleural biopsy specimens of cases negative for malignancy were obtained and used as control samples for immunohistochemical expression of osteopontin. The study was approved by the St Luigi Hospital Institutional Review Board. All samples were made anonymous by a staff member of the pathology department not involved in the study. None of the researchers who managed the tissues and clinicopathologic features had access to disclosed clinical data. Immunohistochemical Analysis For immunohistochemical analysis, 5-µm-thick paraffin sections of each case were collected onto charged slides, deparaffinized, and rehydrated in water. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 minutes. Slides were washed and incubated with a blocking reagent (1% bovine serum albumin in tris(hydroxymethyl) aminomethane-buffered saline) for 10 minutes at room temperature to block nonspecific antibody reactions and then reincubated for 40 minutes at room temperature with antihuman osteopontin polyclonal antibody (Sigma Aldrich, Milan, Italy). After a prolonged wash, the slides were incubated with biotin-free polymer preconjugated with goat antirabbit and peroxidase (En Vision Plus antirabbit-hrp system, DakoCytomation, Glostrup, Denmark) at room temperature for 30 minutes. The reaction product was developed with 3-39-diaminobenzidine tetrahydrochloride (DakoCytomation) as the chromogen. The specificity was validated in serial negative control sections by omitting the primary antibody for each immunohistochemical run. The internal reference control was represented by macrophages whenever present. Immunohistochemical Scoring System Immunohistochemical findings were evaluated by two of us (S.C. and E.B.) and cases with contradictory scores were reevaluated jointly and consensus was reached. Osteopontin immunostaining was observed with different intensity in the cytoplasm of mesothelial neoplastic cells and with a different extent of distribution in the tumor. All cases were analyzed by using a semiquantitative histologic scoring (HScore) method, as described in the literature. 20 Briefly, immunostaining intensity of each case was semiquantitatively scored as follows: 0, none; 1, weak; 2, moderate; and 3, intense, referring to the strong intensity of intratumoral macrophages. In addition, the percentage of positive neoplastic cells was evaluated. For each case, a value designed HScore was obtained by multiplying each intensity with the corresponding percentage of positive cells [HScore = Σ(I PC), where I and PC represent intensity and percentage of cells, respectively], obtaining final resulting score values ranging from 0 to 300. Am J Clin Pathol 2008;130: DOI: /TWCQV536WWRNEU51 59

3 Cappia et al / Prognostic Role of Osteopontin in Mesothelioma Statistical Analysis The Mann-Whitney U and the χ 2 tests were used to analyze differential HScore values of osteopontin expression between groups. The median value of the HScore of the pooled LS and SS groups was calculated at 145 and used as a cutoff to select high ( 145) and low (<145) osteopontin-expressing tumors. Survival analysis comparing high with low osteopontin-expressing tumors was performed by Kaplan- Meier curves and the significance verified by log-rank test. Univariate and multivariate analyses of survival were carried out by means of Cox proportional hazards regression modeling. Results were considered statistically significant if the P value was less than.05. Statistical analysis was performed by using Statistica 7.1 software (StatSoft, Tulsa, OK). Results Clinicopathologic Data In the LS group of 32 MPM cases, all subjects survived more than 24 months, with a range of 24.5 to 69.5 months (median, 37.9 months). In this group, the male/female ratio was 20:12, and the median age at diagnosis was 65 years. An asbestos exposure history was identified in 21 (66%) of 32 patients, and the duration of exposure ranged from 2 to 48 years (mean ± SD, 15.9 ± 12.9 years). At thoracoscopy, the visceral pleura was involved by the tumor in 23 cases (72%). Histologically, all but 3 tumors were epithelioid, and the remaining were biphasic MPMs. The mean proliferative index of these cases, as detected by Ki-67 immunostaining, was 12.5%, consistent with previous data. 21 A positive smoking history was reported in 15 patients, and symptoms were distributed as follows: cough, 14 patients; dyspnea, 22; and chest pain, 12. Weight loss of 5% or more was documented in 3 patients; performance status was less than 80% in 4; a platelet count of /µl ( /L) or less was detected in 15 cases; the time from symptoms to diagnosis was less than 4 months in 12 cases. Seven patients received one or more courses of cisplatin-based chemotherapy. The SS group of MPM cases had an overall median survival time of 11.4 months (range, 1-20 months); the male/ female ratio was 50:19, and this group had almost overlapping clinicopathologic features with the matched cases. The median age was 64 years. Information on possible asbestos exposure was available for 51 (74%) of 69 patients; all but 1 were exposed, with a duration of exposure ranging from 1 to 74 years (mean ± SD, 21.2 ± 14.8 years). At thoracoscopy, the visceral pleura was involved by the tumor in 54 (78%) of 69 cases; epithelioid and biphasic histologic features were present in 63 and 6 cases, respectively. The mean proliferative index as detected by Ki-67 immunostaining was 41.2%, consistent with the literature data. 21 Smoking history was positive in 32 of 69 patients. Symptoms were present as follows: cough, 22; dyspnea, 47; and chest pain, 35. Weight loss of 5% or more was documented in 9 cases, performance status was less than 80% in 17, the platelet count was /µl ( /L) or less in 39, and time to diagnosis was less than 4 months in 37. Cisplatin-based chemotherapy was administered to 19 of 69 patients. The consecutive MPM group (CC group) included 60 men and 21 women with a median age of 61 years at diagnosis. Histologically, there were 46 epithelioid, 16 biphasic, and 19 sarcomatoid MPMs. Nonmalignant control cases were represented by cases of normal pleura (spontaneous pneumothorax, 4 cases) and reactive mesothelial hyperplasia (in chronic pleuritis, 4 cases). Immunohistochemical Staining for Osteopontin Osteopontin reactivity was specifically localized in internal control cells (ie, macrophages). No immunoreactivity was observed in normal mesothelial cells obtained from patients with pneumothorax or in the surrounding stroma. Conversely, hyperplastic or reactive mesothelial cells in the cases of nonmalignant chronic pleuritis had weak immunoreactivity. Occasional mesenchymal and inflammatory cells in the extracellular matrix were positive for osteopontin. Osteopontin expression was observed in almost all MPM cases. Neoplastic cells had variable patterns of cytoplasmic reactivity, diffuse granular or as perinuclear spots, with intensity ranging from weak (score 1) to intense (score 3) zimage 1z. The percentage of distribution varied from 1% to 98%; therefore, the HScore values obtained ranged from 1 to 297. In many samples, osteopontin reactivity appeared to be restricted to small, very strong cytoplasmic spots, immediately adjacent to the nucleus. This staining pattern probably corresponds to osteopontin protein in the Golgi apparatus and/or secretory vesicles. 22 In the LS group, the distribution of HScore values of osteopontin expression varied from 5 to 250 (median, 50), whereas in the SS group, HScore values varied from 30 to 297 (median, 180). The difference was statistically significant (P <.0001; Mann-Whitney U) zfigure 1Az. No statistically significant difference (P =.3817; Mann-Whitney U) was found between the pooled LS and SS groups, in which the distribution of HScore values ranged from 5 to 297 (median, 145), and the CC group, ranging from 1 to 285 (median, 132). The two groups were therefore statistically comparable zfigure 1Bz Correlation Analysis of Osteopontin Expression and Clinical and Pathologic Parameters In the LS and SS groups, osteopontin expression had no significant correlation with sex, age, asbestos exposure, thoracoscopic stage, or histologic type. In addition, no correlation with smoking history, symptoms, platelet count, or chemotherapy was observed. In contrast, a significant correlation 60 Am J Clin Pathol 2008;130: DOI: /TWCQV536WWRNEU51

4 Anatomic Pathology / Original Article A C B zimage 1z Osteopontin expression in malignant pleural mesothelioma (MPM). A and B, High histologic scoring (HScore) for immunohistochemical expression of osteopontin was generally observed in short-survival cases of MPM (A and B, immunoperoxidase, 400). C, Most long-survival cases had low HScore osteopontin immunoreactivity, with a lower percentage of positive tumor cells and weaker immunostaining compared with short-survival cases of MPM (immunoperoxidase, 200) LS SS HScore OPN Expression H-Score OPN Expression prognostic factors (P < and P =.02, respectively), whereas no other variables correlated with survival ztable 1z. In multivariate analysis, however, only osteopontin retained significance as an independent predictor of survival. zfigure 2z shows Kaplan-Meier survival curves of patients according to low and high osteopontin expression (log-rank test). B A Discussion LS + SS CC zfigure 1z Distribution of osteopontin (OPN) immunohistochemical histologic scoring (HScore) values in malignant pleural mesothelioma (MPM). A, OPN HScore values were lower in long-term survival (LS) than in short-term survival (SS) cases (P <.0001; Mann-Whitney U). B, OPN HScore values in the pooled LS and SS (LS + SS) cases were comparable to the consecutive MPM group (CC) (P =.3817; Mann-Whitney U). Boxes indicate the range (25th-75th percentiles); whiskers, the major and minor values; and lines in the boxes, median values. between osteopontin expression and weight loss (P =.04; χ2), performance status (P =.006; χ2), and time from symptoms to diagnosis (P =.004; χ2) was recorded. Survival Analysis of Patients With MPM In univariate analyses comparing the LS and SS groups, osteopontin expression and performance status were significant In this study, it was confirmed that osteopontin is expressed in MPM and, for the first time, it was found that its reactivity is significantly reduced in long-term surviving patients. In addition, as previously reported,17 osteopontin expression was also detected in reactive or hyperplastic mesothelial lesions, limiting the proposed diagnostic role in MPM,16 at least at the tissue level. Osteopontin was up-regulated in asbestos-induced tumors developed in a rat model of asbestos carcinogenesis and in cells exposed to asbestos in vitro.23 Recently, osteopontin was proposed as a serum marker of MPM in asbestos-exposed people.16 Based on these observations, a study was designed to assess possible differential osteopontin expression levels in MPM cases having different outcomes. Long survival is a rare event in MPM, accounting for fewer than 18% of the approximately 400 cases seen at St Luigi Hospital. This figure is in agreement with literature data.24,25 Therefore, in this study, data for all MPM cases with long survival were retained, whereas data for only a random sample of comparable short-term survivors were included. 61 Am J Clin Pathol 2008;130: DOI: /TWCQV536WWRNEU51

5 Cappia et al / Prognostic Role of Osteopontin in Mesothelioma ztable 1z Univariate and Multivariate Analysis for Cases of Malignant Pleural Mesothelioma * Univariate Analysis Multivariate Analysis No. of Cases MS HR (Range) P HR (Range) P Sex 0.77 ( ) ( ).62 M F Age (y) 1.02 ( ) ( ).92 < > Smoking 0.96 ( ) ( ).37 No Yes Asbestos exposure Level 1.01 ( ) ( ).71 No Low Intermediate High Duration (y) 0.85 ( ) ( ).22 < Cough 0.93 ( ) ( ).51 No Yes Chest pain 1.2 ( ) ( ).3 No Yes Dyspnea 1.3 ( ) ( ).15 No Yes Weight loss (%) 1.24 ( ) ( ).99 < Performance status (%) 0.55 ( ) ( ).59 < Platelet count ( 10 3 /µl) 0.83 ( ) ( ) > Thoracoscopy 0.97 ( ) ( ).09 Vegetation Plaque Histologic findings 1.02 ( ) ( ).51 Epithelioid Biphasic Chemotherapy 0.93 ( ) ( ).89 No Yes Osteopontin HScore 3.67 ( ) < ( ) < < HR, hazard ratio; HScore, histologic scoring; MS, median survival. * Osteopontin expression is an independent prognostic marker in malignant pleural mesothelioma. Platelet values are given in conventional units; to convert to Système International units ( 10 9 /L), multiply by 1.0. The distinction between long- and short-term survival of patients with MPM was arbitrarily set using a cutoff of 24 months, according to the reported median survival time of 4 to 12 months, with few patients surviving longer than 2 years. 3 We retrospectively identified 32 cases from a long period with survival longer than 24 months. Unfortunately, serum samples were not available for circulating osteopontin measurements, but paraffin blocks from pleural biopsies were retrieved. Each long-surviving MPM case was matched with at least 2 cases of MPMs with similar clinicopathologic characteristics, except for the outcome that followed the expected fatal course within 20 months (mean, 11.1 months). In this latter group, osteopontin expression levels were significantly higher than those of the LS group (P <.0001), a difference maintained also at multivariate analysis, indicating an independent prognostic role of osteopontin in MPM. The 62 Am J Clin Pathol 2008;130: DOI: /TWCQV536WWRNEU51

6 Anatomic Pathology / Original Article Cumulative Proportion Surviving High OPN Time (mo) Low OPN zfigure 2z Cumulative proportion surviving according to osteopontin (OPN) expression. Kaplan-Meier survival curves of patients according to low and high OPN expression (P <.0001; log-rank test). The median histologic scoring (HScore) value of 145 was selected as the cutoff. prevalence of osteopontin expression was also independently assessed in a consecutive series of 81 MPM cases diagnosed by pleural biopsies at St Luigi Hospital, University of Turin, from 2005 to Osteopontin immunoreactivity was detected in the vast majority of cases with focal or low reactivity (HScore <30) in 7 (9%) of 81 cases only and a diffuse and/or strong reactivity in the remaining cases. The prognostic role of osteopontin demonstrated in MPMs is independent of other prognostic factors, such as sex, age, stage, and histologic type. 26 Indeed, the majority of cases in the present study were epithelioid MPMs, which are known to have a better prognosis than the sarcomatoid type. However, within such a subtype, heterogeneity of outcome exists, and osteopontin tissue levels were significantly different in the LS vs SS groups. The tumor proliferative fraction was also found to be of prognostic value; high Ki-67 values were associated with shorter survival. 21 In this study, immunohistochemical analysis was the method used to detect osteopontin, based on previous reports that showed good correlation of immunohistochemical osteopontin expression levels with those of Western blot analysis, enzyme immunoassay, and corresponding messenger RNA analysis. 11,12,27 The osteopontin pattern of distribution was in the form of diffuse granular cytoplasmic reactivity and, more rarely, of paranuclear dots in the Golgi area. 22 This latter pattern was restricted to MPM cases with a poor prognosis (10/69 [14%]). Because osteopontin requires phosphorylation and glycosylation in the Golgi apparatus before being secreted from the cell, the observed punctuate osteopontin staining may reflect increased production and processing of the protein. The assessment of osteopontin immunohistochemical findings by the HScore method 20 takes into account the intensity and percentage of distribution of positive cells. Therefore, with respect to the standard evaluation of immunohistochemical results by a 4-tier score (0-3), it gives a more accurate and homogeneous assessment of the protein expression levels in individual cases. In fact, it allows combining heterogeneous patterns of distribution (eg, diffuse, weak signal and limited areas of strong reactivity), thus reducing the risk of underestimating or overestimating the values for overall protein expression. Increased osteopontin levels have been detected in the plasma or serum derived from patients with a number of solid neoplasms, including ovarian, 12 head and neck, 28 prostate, 29 colon, breast, and lung 30 cancers. Samples from patients with multiple myeloma 31 and MPM 16,18,32 were also found to contain high levels of circulating osteopontin. In MPM, osteopontin serum measurements were shown to be of low diagnostic usefulness owing to the lack of specificity but were of potential value as a prognostic marker together with mesothelin, another product of mesothelial cells. 32 A major limitation of circulating osteopontin measurements is that blood measurements are referred to total osteopontin and do not distinguish osteopontin variants that can be produced by cancer cells, as opposed to normal noncancerous cells. 33 At least 3 splice variants of osteopontin have been identified so far, lacking exon 5 or Osteopontin expression was statistically correlated with a subgroup of long-surviving patients with MPM, as opposed to patients with short survival. These data indicate that increased osteopontin expression levels may be a predictive marker of survival at the time of MPM diagnosis. As a consequence, more aggressive initial therapy or treatments targeting the osteopontin protein with antibodies 35,36 and/or osteopontin receptors by cytotoxic and immunotherapeutic strategies 37,38 may be envisaged. In addition, it is proposed that osteopontin expression be considered as a stratification factor for future trials assessing different treatment modalities in MPM. From the 1 Department of Clinical and Biological Sciences, University of Torino at St Luigi Hospital, Torino, Italy; and 2 Unit of Cancer Epidemiology, CeRMS and CPO-Piemonte, University of Torino, Torino. Supported by grants from the Italian Ministry of Research, Rome (Giorgio V. Scagliotti and Mauro Papotti) and the Regione Piemonte (Progetto Ricerca Sanitaria Finalizzata 2007 DRG n del to Mauro Papotti). Address reprint requests to Dr Mauro Papotti: Dept of Clinical and Biological Sciences, University of Turin, Division of Pathology, St Luigi Hospital, Regione Gonzole 10, Orbassano, Torino, Italy. Acknowledgments: We are grateful to Gianni Bussolati, MD, FRCPath, Piero Borasio, MD, Franco Merletti, MD, and Marco Volante, MD, PhD, University of Turin, for their helpful suggestions. Am J Clin Pathol 2008;130: DOI: /TWCQV536WWRNEU51 63

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